Summary Influenza A viruses (IAV) are constantly changing. This change occurs rapidly, on a similar time scale to influenza epidemics. For this reason, the epidemiology and evolution of IAV are closely linked, and an in depth understanding of viral evolution is critical for public health efforts aimed at controlling influenza. We are working to understand the mechanisms of IAV genomic diversification, which underlie its evolution. The current proposal focuses on reassortment, the process by which influenza and other viruses with segmented genomes exchange gene segments. The potential for reassortment to purge the viral genome of deleterious mutations and bring together multiple beneficial changes makes it a powerful catalyst of viral evolution. Reassortant viruses that derive gene segments from human and avian or swine-adapted IAV can furthermore overcome host restrictions to cause zoonoses or pandemics. Indeed, reassortment enabled all four IAV pandemics of the last century. However, reassortment between heterologous IAVs is subject to strong constraints, due to the potential for incompatibility among divergent viral proteins or RNAs. Herein, we propose to measure the impact on reassortment efficiency of sequence divergence within viral RNA packaging signals. In this way, we will test the hypothesis that selective genome packaging limits reassortment. We expect that the severity of the restriction on reassortment will correlate inversely with sequence identity within packaging signal regions and that, for this reason, certain segment combinations will be more likely to arise than others. We furthermore predict that the genotypes that emerge from heterologous reassortment will reflect the physical interactions among viral RNA segments during virion assembly. We will therefore use our data to construct testable models of how the eight segments are organized within the virion. This approach brings a novel methodology to the perennially difficult problem of how the genome is packaged into viral particles. Finally, to gauge the importance of packaging signal mismatch relative to protein mismatch, we will evaluate the impact of sequence divergence within viral protein coding sequences on reassortment outcomes. Ultimately, we aim to define the conditions in nature that are permissive for reassortment and the factors that determine the efficiency of reassortment. This knowledge will significantly advance our understanding of IAV evolution and the mechanisms that shape the emergence of zoonotic and pandemic IAVs.